In previous studies, we have shown that RSV replication in A549 cells (a type II alveolar epithelial cell line) induces a dose- and time-dependent release of IL-8. We have subsequently investigated the mechanisms of IL-8 induction. By RT-PCR, the IL-8 mRNA, barely detectable in control cells, was strongly induced by RSV infection; the IL-8 mRNA increase was due to an enhanced transcriptional initiation, as shown by nuclear “run-on” transcription rate analysis. A549 cells were transiently transfected with plasmids containing either wild type or deleted mutants of IL-8 promoter, linked to a luciferase reporter gene, and were infected with RSV. The results showed that a region between -162 and +33 of the promoter is sufficient to induce transcription of the IL-8 gene, after RSV infection. Mutations in the NF-kB binding site, comprised in this region of the promoter, abolished the IL-8 gene induction by RSV. The TNF-responsive element (TNFRE) is a region of IL-8 promoter sufficient to confer induction of the IL-8 promoter to different inflammatory stimuli. Electrophoretic mobility shift assays (EMSA), using a32 P labeled TNFRE as a probe, showed increased DNA-binding activity after RSV infection. To characterize the protein-DNA complexes binding specificity, we used wild type TNFRE or mutated TNFRE for NF-IL6 or NF-kB binding sites as competitors in the EMSA. We also performed gel mobility supershift assays, adding an anti p65 (RelA) antibody to infected cells nuclear extracts. The results indicated that RSV-induced DNA binding proteins belong to the NF-kB family and contain the RelA subunit. An increase of nuclear RelA, after RSV infection, was also demonstrated by Western Blot. Although the precise mechanism of RelA activation will require further study, by immunofluorescence we observed translocation of RelA from the cytoplasmic to the nuclear compartment. These studies establish RelA activation as one mechanism for RSV-induced inflammation in airway epithelium. Supported by NIAID AI15939, Texas ARP/ATP.